The goal of this project is the development of an innovative platform for discovery of small molecule chemokine inhibitors. Chemokines direct cell migration primarily by binding and activating a family of G protein-coupled receptors that are post-translationally modified by tyrosine sulfation. Sulfation is required for chemokine function, suggesting that receptor sulfotyrosines participate directly in specific binding of the chemokine ligand. We recently revealed the molecular basis for sulfotyrosine recognition in the chemokine family by solving the NMR structure of a complex between the chemokine SDF1/CXCL12 and a sulfotyrosine-containing fragment of its receptor CXCR4. The N-terminal extracellular domain of CXCR4 contains three potential sulfation sites, and each sulfotyrosine occupies a unique cleft on the CXC12 surface. We hypothesize that because sulfotyrosine recognition sites are essential for high affinity receptor binding, each one represents a target for inhibition using small molecules directed at the chemokine ligand. In specific aim 1 we propose to define additional sulfotyrosine recognition sites on the CXCL12 surface and use a combination of high-throughput docking calculations, validation of site-specific ligand binding by 2D NMR, and functional assays to test for chemokine inhibition. We will refine our in silico screening methods in aim 2 using the structure of the CXCL12/CXCR4 complex as a test case for evaluation of flexible side chain docking and fragment screening against the ensemble of NMR conformers. Hit-to-lead and lead optimization will be performed for affinity and selectivity in specific aim 3 using SAR analysis and by designing and synthesizing novel compounds that combine ligands from adjacent sulfotyrosine recognition sites. Compounds that bind CXCL12 and function as potent and selective inhibitors in cell-based chemokine assays will be made available to existing collaborators for preclinical studies in animals. To assess the broader potential of sulfotyrosine- directed inhibition, we will develop a streamlined approach to site identification in specific aim 4 and apply our screening method to a representative set of chemokine targets. Structure-based drug discovery guided by sulfotyrosine recognition is a conceptual advance that can be applied to all 50 members of the chemokine family and other complexes that require this protein modification to function in the extracellular space. New treatments for inflammatory and autoimmune disease or cancer that emerge from application of this strategy will have a major impact on human health.